Title

Author

Date of Award

2008

Degree Name

Biological Sciences

College

College of Science

Type of Degree

M.S.

Document Type

Thesis

First Advisor

Andrew Rogerson

Second Advisor

Wendy Trzyna

Third Advisor

Philippe Georgel

Abstract

Amoebae are important heterotrophic protists (protozoa) and this study focuses on three unusual forms from the marine environment. Amoebae are ubiquitous and play an important role as consumers in microbial communities. A common coastal ctenophore (Mnemiopsis sp.) is known to harbor an undescribed naked amoeba on the comb plate surface. The nature of the symbiotic association is unknown although electron microscopy suggests these micrograzers are degrading comb plates (Moss et al., 2001). A second amoeba isolate from mangrove waters is new to science by virtue of its distinctive trophic form that does not conform to any described species. A third isolate, Acanthamoeba spp. is unusual because it was isolated from offshore marine waters despite the fact it is normally found in soil. On rare occasions acanthamoebae invade the cornea and cause Amoebic Keratitis (AK). Characterization of these amoebae involved characterizing three important diagnostic features including morphology (size, form and locomotion), physiology (salinity tolerance) and molecular analysis. Salinity tolerance experiments (0g/l to 50g/l) were conducted to better understand the origin of the isolates while the molecular analysis was conducted with an aim to determine phylogeny of the isolates. The Acanthamoeba marine isolate was compared to non-marine strains isolated from other ‘unusual’ environments including chlorinated tap water, acidic soil (pH4) and marine fish scale mucus. These were included to determine their genotypes and to explore a suggested possible link between survivability in extreme habitats and pathogenicity (Booton et al., 2004). Characterizing the ctenophore amoeba included all three diagnostic features while studies on the second amoeba isolate involved physiological studies (salinity tolerance) and molecular analysis (ribotyping) based on small subunit (SSU) ribosomal RNA gene (Medlin et al., 1988; Smirnov et al., 2007). Studies on acanthamoebae included physiological studies and genotyping based on the 18S small subunit ribosomal RNA gene (Booton et al., 2004). The ctenophore amoeba had an optimum generation time at 10g/l salt (7.9 hours) suggesting that this is an estuarine amoeba although cells survived up to 50g/l. The amoeba from mangrove waters had its optimum generation time at 20g/l (34 hours) and amoebae survived up to 50g/l. The "marine" Acanthamoeba grew best at 15g/l salt suggesting that this may be an unusual strain with a unique genotype. Phylogenetic analyses showed that the marine strain was a T3 designate while tap water strains and acid tolerant acanthamoebae were T4 strains. The acid tolerant Acanthamoeba strain closely resembled A. castellanii previously associated with AK infections (98% bootstrap value; 0.2% dissimilarity). Acanthamoeba strain from fish mucus closely resembled T5 designates (78% bootstrap value; 5% dissimilarity). There was no notable trend seen with acanthamoebae genotypes and association with "extreme" environments. However, a T3 Acanthamoeba designate has previously been reported to tolerate salinity as high as 32g/l (Sawyer 1970, 1971). Interestingly, all three unusual amoebae isolates showed optimum growth between 10g/l and 20g/l indicating that amoebae, in general, may prefer this low salinity to save on energetic costs involved in expelling water via their contractile vacuoles.